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Positive Nasal Allergen Provocation Test and Nasal Specific-IgE to Grass Pollen in Patients with Idiopathic Rhinitis
S314 Abstracts
1225
Activation of Protein Kinase D1 (PKD1) in Mast Cells by TLR, Kit, and FcepsilonRI ligands T. R. Murphy, H. R. Katz; Harvard Med. Sch./Brigham & Women’s Hosp., Boston, MA. RATIONALE: Little is known about the serine/threonine kinase PKD1 in mast cells. We sought to define ligands that activate PKD1 in mast cells and to begin to address the contributions of PKD1 to mast cell activation. METHODS: PKD1 in mouse bone marrow-derived mast cells (BMMC) was detected with RT-PCR and immunoblotting. Activation of PKD1 was measured by immunoblotting directed to the autophosphorylation site of PKD1 (pS916). MCP-1 and b-hexosaminidase were quantified by ELISA and enzymatic activity, respectively. RESULTS: BMMC contained PKD1 mRNA and immunoreactive PKD1. The TLR2 ligand Pam3CSK4 induced MyD88-dependent activation of PKD1 that was inhibited by the PKD1/PKC inhibitor Go¨ 6976 but not by the PKC inhibitor Bim I. Go¨ 6976 also inhibited Pam3CSK4-induced release of MCP-1 at concentrations comparable to those that inhibited PKD1 activation. Activation of BMMC by SCF or by crosslinking IgE bound to FceRI also induced activation of PKD1 and release of MCP-1, and each response was inhibited by Go¨ 6976 but not by Bim I. In contrast, both agents inhibited exocytosis of b-hexosaminidase induced by SCF or crosslinked FceRI. CONCLUSIONS: Our findings establish that stimuli representing innate, adaptive, and growth factor pathways activate PKD1 in mast cells. Inhibition of PKD1 activation was associated with suppression of a mediator produced after gene induction, whereas PKCs may play a more predominant role in regulating exocytosis. Furthermore, our data represent the first demonstration in any cell type that a TLR ligand activates PKD1, suggesting a regulatory role for this enzyme in innate immune and inflammatory processes. Funding: NIH
1226
TUESDAY
CysLT2 Receptors Negatively Regulate CysLT1 Receptor Functions on Mast Cells Through Heterodimerization J. A. Boyce, Y. Jiang; Brigham and Women’s Hospital, Boston, MA. RATIONALE: Cysteinyl leukotrienes (cys-LTs) are potent stimulants of signaling events and proliferation of mast cells (MCs). Despite the presence of CysLT2 receptors on MCs, these responses can be largely blocked by antagonists of CysLT1 receptors. METHODS: We studied functional responses of human MCs to exogenous cys-LTs following short-hairpin (sh)RNA-based manipulation of CysLT1 and CysLT2 receptor expression. In parallel, we studied these responses in MCs from mice lacking each receptor. We used biochemical and imaging techniques to identify receptor-receptor interactions. RESULTS: CysLT1 and CysLT2 receptors co-precipitate from extracts of a human MC line, LAD2, and from primary cord blood-derived human MCs (hMCs), and strikingly co-localized at the surfaces of LAD2 cells and hMCs, as determined by confocal imaging. Antibody-based fluorescent lifetime imaging (FLIM) confirmed the formation of heterodimers based on fluorescence energy transfer (FRET). Knock-down of CysLT1 receptors using shRNA abrogated both cys-LT-dependent proliferation and cytokine generation, whereas cells subjected to CysLT2 knock-down displayed enhanced responses; these were abrogated by treatment of the cells with the CysLT1 receptor antagonist, MK571. Cys-LT-mediated c-Kit transactivation, ERK phosphorylation, and cytokine generation were abrogated and enhanced, respectively, by the lack of CysLT1 and CysLT2 receptors in mouse mBMMCs. CONCLUSIONS: CysLT2 receptors negatively regulate cys-LT-induced MC functions, possibly through formation of heterodimers that dampen signaling. Funding: NIH/NIAID
J ALLERGY CLIN IMMUNOL JANUARY 2007
1227
The Ability of the Nose to Warm and Humidify Inspired Air is Heritable J. Pinto, A. Sahin-Yilmaz, F. Baroody, M. deTineo, S. Elwany, C. Ober, R. M. Naclerio; The University of Chicago, Chicago, IL. RATIONALE: In our previous studies on nasal air conditioning, we observed a large variability among the ability of individuals to condition inspired air. Although we previously investigated different parameters like age, sex, nasal mucosal temperature, pulse, blood pressure and nasal volume, we have been unable to explain this variability. In this study we hypothesized that heredity contributes to the differences in the nasal conditioning capacity of individuals. METHODS: We performed a prospective study on 47 sibling pairs, ages 18 to 35 years. Cold dry air was delivered to the nose at 3 different flow rates and the total water gradient was calculated to reflect the nasal conditioning capacity. RESULTS: We found a highly significant intraclass correlation of 0.53 (p<0.0001) between sibling pairs for the total water gradient CONCLUSIONS: These results suggest that there is a genetic basis for nasal conditioning.
1228
Positive Nasal Allergen Provocation Test and Nasal Specific-IgE to Grass Pollen in Patients with Idiopathic Rhinitis C. Rondon1, J. J. Romero1, S. Lopez2, R. R-Pena2, J. L. Rodriguez-Bada2, P. Chaves2, I. Don˜a1, M. Blanca1; 1Allergy Service, Carlos Haya Hospital, Ma´laga, SPAIN, 2Research Laboratory, Fundacion IMABIS-Carlos Haya Hospital, Ma´laga, SPAIN. RATIONALE: Recently the concept of a localized mucosal allergy disease in absence of atopy has been proposed, usually in patients with persistent rhinitis. In this study we investigated the response to nasal allergen provocation test to grass pollen (NAPT-GP) and the presence of nasal pollen-specific IgE in idiopathic rhinitis patients who referred symptoms only during spring (SIR). METHODS: 12 patients with SIR, 24 patients with intermittent allergic rhinitis to grass pollen (IAR), and 20 healthy controls (HC) were studied to test the response to NAPT-GP, the presence of nasal grass, olive and parietaria pollen-specific IgE (CAP-FEIA), ECP, and leukocyte phenotype by flow cytometry with monoclonal antibodies (CD16-FITC, CD8-FITC, CD4-PE, CD33-PE, CD3-PerCP, and CD45-APC) in nasal lavage. RESULTS: In the SIR group the 75% showed a positive NPT-GP, and in two of these (22%) we detected nasal pollen-specific IgE, one to grass and olive, and the other one to grass and parietaria pollen. These patients showed a similar leukocyte pattern to the IAR group (eosinophils > 50%, neutrophils < 13% and total lymphocytes 1%). NAPT-GP was positive in all IAR subjects, and negative in HC. Nasal ECP levels were significantly higher in patients with IAR compared with SIR and HC (p < 0.05). CONCLUSIONS: These data sustain the hypothesis that some idiopathic rhinitis patients may present a local intermittent allergy rhinitis in absence of atopy. Further studies are needed to evaluate the influence of other allergens.
1225
Activation of Protein Kinase D1 (PKD1) in Mast Cells by TLR, Kit, and FcepsilonRI ligands T. R. Murphy, H. R. Katz; Harvard Med. Sch./Brigham & Women’s Hosp., Boston, MA. RATIONALE: Little is known about the serine/threonine kinase PKD1 in mast cells. We sought to define ligands that activate PKD1 in mast cells and to begin to address the contributions of PKD1 to mast cell activation. METHODS: PKD1 in mouse bone marrow-derived mast cells (BMMC) was detected with RT-PCR and immunoblotting. Activation of PKD1 was measured by immunoblotting directed to the autophosphorylation site of PKD1 (pS916). MCP-1 and b-hexosaminidase were quantified by ELISA and enzymatic activity, respectively. RESULTS: BMMC contained PKD1 mRNA and immunoreactive PKD1. The TLR2 ligand Pam3CSK4 induced MyD88-dependent activation of PKD1 that was inhibited by the PKD1/PKC inhibitor Go¨ 6976 but not by the PKC inhibitor Bim I. Go¨ 6976 also inhibited Pam3CSK4-induced release of MCP-1 at concentrations comparable to those that inhibited PKD1 activation. Activation of BMMC by SCF or by crosslinking IgE bound to FceRI also induced activation of PKD1 and release of MCP-1, and each response was inhibited by Go¨ 6976 but not by Bim I. In contrast, both agents inhibited exocytosis of b-hexosaminidase induced by SCF or crosslinked FceRI. CONCLUSIONS: Our findings establish that stimuli representing innate, adaptive, and growth factor pathways activate PKD1 in mast cells. Inhibition of PKD1 activation was associated with suppression of a mediator produced after gene induction, whereas PKCs may play a more predominant role in regulating exocytosis. Furthermore, our data represent the first demonstration in any cell type that a TLR ligand activates PKD1, suggesting a regulatory role for this enzyme in innate immune and inflammatory processes. Funding: NIH
1226
TUESDAY
CysLT2 Receptors Negatively Regulate CysLT1 Receptor Functions on Mast Cells Through Heterodimerization J. A. Boyce, Y. Jiang; Brigham and Women’s Hospital, Boston, MA. RATIONALE: Cysteinyl leukotrienes (cys-LTs) are potent stimulants of signaling events and proliferation of mast cells (MCs). Despite the presence of CysLT2 receptors on MCs, these responses can be largely blocked by antagonists of CysLT1 receptors. METHODS: We studied functional responses of human MCs to exogenous cys-LTs following short-hairpin (sh)RNA-based manipulation of CysLT1 and CysLT2 receptor expression. In parallel, we studied these responses in MCs from mice lacking each receptor. We used biochemical and imaging techniques to identify receptor-receptor interactions. RESULTS: CysLT1 and CysLT2 receptors co-precipitate from extracts of a human MC line, LAD2, and from primary cord blood-derived human MCs (hMCs), and strikingly co-localized at the surfaces of LAD2 cells and hMCs, as determined by confocal imaging. Antibody-based fluorescent lifetime imaging (FLIM) confirmed the formation of heterodimers based on fluorescence energy transfer (FRET). Knock-down of CysLT1 receptors using shRNA abrogated both cys-LT-dependent proliferation and cytokine generation, whereas cells subjected to CysLT2 knock-down displayed enhanced responses; these were abrogated by treatment of the cells with the CysLT1 receptor antagonist, MK571. Cys-LT-mediated c-Kit transactivation, ERK phosphorylation, and cytokine generation were abrogated and enhanced, respectively, by the lack of CysLT1 and CysLT2 receptors in mouse mBMMCs. CONCLUSIONS: CysLT2 receptors negatively regulate cys-LT-induced MC functions, possibly through formation of heterodimers that dampen signaling. Funding: NIH/NIAID
J ALLERGY CLIN IMMUNOL JANUARY 2007
1227
The Ability of the Nose to Warm and Humidify Inspired Air is Heritable J. Pinto, A. Sahin-Yilmaz, F. Baroody, M. deTineo, S. Elwany, C. Ober, R. M. Naclerio; The University of Chicago, Chicago, IL. RATIONALE: In our previous studies on nasal air conditioning, we observed a large variability among the ability of individuals to condition inspired air. Although we previously investigated different parameters like age, sex, nasal mucosal temperature, pulse, blood pressure and nasal volume, we have been unable to explain this variability. In this study we hypothesized that heredity contributes to the differences in the nasal conditioning capacity of individuals. METHODS: We performed a prospective study on 47 sibling pairs, ages 18 to 35 years. Cold dry air was delivered to the nose at 3 different flow rates and the total water gradient was calculated to reflect the nasal conditioning capacity. RESULTS: We found a highly significant intraclass correlation of 0.53 (p<0.0001) between sibling pairs for the total water gradient CONCLUSIONS: These results suggest that there is a genetic basis for nasal conditioning.
1228
Positive Nasal Allergen Provocation Test and Nasal Specific-IgE to Grass Pollen in Patients with Idiopathic Rhinitis C. Rondon1, J. J. Romero1, S. Lopez2, R. R-Pena2, J. L. Rodriguez-Bada2, P. Chaves2, I. Don˜a1, M. Blanca1; 1Allergy Service, Carlos Haya Hospital, Ma´laga, SPAIN, 2Research Laboratory, Fundacion IMABIS-Carlos Haya Hospital, Ma´laga, SPAIN. RATIONALE: Recently the concept of a localized mucosal allergy disease in absence of atopy has been proposed, usually in patients with persistent rhinitis. In this study we investigated the response to nasal allergen provocation test to grass pollen (NAPT-GP) and the presence of nasal pollen-specific IgE in idiopathic rhinitis patients who referred symptoms only during spring (SIR). METHODS: 12 patients with SIR, 24 patients with intermittent allergic rhinitis to grass pollen (IAR), and 20 healthy controls (HC) were studied to test the response to NAPT-GP, the presence of nasal grass, olive and parietaria pollen-specific IgE (CAP-FEIA), ECP, and leukocyte phenotype by flow cytometry with monoclonal antibodies (CD16-FITC, CD8-FITC, CD4-PE, CD33-PE, CD3-PerCP, and CD45-APC) in nasal lavage. RESULTS: In the SIR group the 75% showed a positive NPT-GP, and in two of these (22%) we detected nasal pollen-specific IgE, one to grass and olive, and the other one to grass and parietaria pollen. These patients showed a similar leukocyte pattern to the IAR group (eosinophils > 50%, neutrophils < 13% and total lymphocytes 1%). NAPT-GP was positive in all IAR subjects, and negative in HC. Nasal ECP levels were significantly higher in patients with IAR compared with SIR and HC (p < 0.05). CONCLUSIONS: These data sustain the hypothesis that some idiopathic rhinitis patients may present a local intermittent allergy rhinitis in absence of atopy. Further studies are needed to evaluate the influence of other allergens.